The Section on Cellular Biophotonics, of the Laboratory of Molecular Physiology, NIAAA, was established in April 2003. The principle aim of this section is to use imaging techniques to study how protein complexes, with special emphasis on complexes comprised of integral membrane proteins, are formed and maintained in living cells. Membrane proteins, such as receptors, channels, and their regulatory partners, are typically synthesized by ribosome?s associated with the endoplasmic reticulum, but ultimately function at the cell surface. Thus, the logistics of their production, assembly, transport, and exocytotic insertion into the cell surface is key to understanding both normal physiological function, and abnormal physiological states associated with human maladies. Furthermore, the delivery of membrane protein complexes to cell surface micro-domains must involve the coordination of exocytotic and endocytotic reactions. Lesions in exocytosis have been tied to some forms of muscular dystrophy, and may also be involved in alcohol induced myopathy. Drs. Srinagesh Koushik , and Christopher Thaler, joined the section in May 2003 as a Research Fellow, and as a postdoctoral IRTA respectively. Dr. Fernando Covian-Nares joined the section in May 2004 as a postdoctoral IRTA. Over the past year we have primarily been involved in assembling and testing a new microscope capable of multi-photon laser scanning imaging, spectral imaging, and time-resolved fluorescence imaging. All three of these imaging modes are operational. We are currently pursuing several research projects that utilized the capabilities of this new imaging system. Research highlights over this past year include: 1. The completion of our study that investigated the validity of using spectral un-mixing for quantifying CFP and YFP concentrations in living cells. We found that spectral un-mixing can yield quantitative results as long as reference spectra are baseline corrected, and fluorescence resonance energy transfer (FRET) is not occurring between donor (CFP) and acceptor (YFP) molecules. 2. We next developed and tested a new spectral un-mixing algorithm that works in the presence and absence of FRET. This new method can be used to measure the intracellular abundance of CFP and YFP tagged molecules regardless of molecular interactions between the tagged proteins, and it can also be used to measure FRET efficiencies in living cells, an indicator of molecular interactions. 3. We have shown that sea urchin eggs have at least two different mechanisms of compensatory endocytosis. We next demonstrated that these cellular mechanisms retrieve specific and unique patches of the cell surface membrane. One of these mechanisms specifically retrieved membranes inserted by calcium triggered exocytosis, while the second mechanism retrieved membrane patches inserted by constitutive exocytosis. Additionally, we have also been involved in two collaborations that are associated with the primary aims of the section. In collaboration with Dr. Stephen Ikedas section, we have been involved in developing and evaluating genetically engineered reagents to monitor G-protein subunit localization and activation in living cells. With Dr. Margaret Davis (in Dr. Dave Lovinger?s Laboratory) we have been involved in developing methods for two-photon uncaging of photactivatible GFP constructs in living cells. This method will be used to study the dynamics of tyrosine-kinase activation and regulation of ion channels on the neuronal cell surface.